Is it possible to re-sequence Index2 on a HiSeq PE dual index run?

[MikeNice]

Background:
Instead of combining 4 50 cycle kits for our HiSeq dual index runs, we typically use standard 200 cycle kits and 1 50 cycle kit to top off up to 4 dual index runs starting on the same day. Further, we split one incorporation mix between FCA and FCB at the start of all our HiSeq runs, and split the second mix at the turn.

Our problem:
On this particular dual index run, the reagents were not topped off at the start of the run, thus the incorporation mix ran dry sometime during the dark cycles, causing index2 to fail. The dilemma here is that simply adding incorporation mix and resuming the run at the beginning of index2 will cause the read to be out of phase since an unknown number of bases were added during the dark cycles before the mix ran dry. Since the index2 primer is grafted to the flowcell we cannot denature the strand and start the read over either.

Any suggestions as to how to save this run? Thanks in advance!

[kcchan]

Calling up tech support might be your best bet at this point. I would guess that a strip and rehyb would still work at this point. The only difference here being the fact that you won't be adding a new primer but instead relying on the grafted flow cell primers.

[HeinKey]

MikeNice,

If you flush your system with high salt to remove the air, and then continue, and repeat the second index?
You first need to tweak the HCS so it will image the dark cycli resulting in 15 cycli imaging instead of 7 dark+8 imaging. How to do this I don't know, this might be impossible...??
If you just repeat it you will miss the first x-cycli which were done in your first dark cycli attempt.

You will have to demultiplex your self since you don't know which cycli will be index cycles. After the P5index I expect the turnaround will complete normally so the reverse read can be sequenced normally.

@kcchan, I am not sure of strip and rehyb are necessary. I think the grafted oligo's are fixed and extended with 1-7 (dark cycle) nucleotides. I expect these extended oligo's to rehyb to the first strands ending up in the same situation as before strip and rehyb.

[MikeNice]

@HeinKey and @kcchan, thank you both so much for your replies.

As HeinKey pointed out, I don't think the strip and rehyb work in this scenario because the index2 primer and therefore unknown number of extended bases are grafted to the flowcell (this is unlike all other reads, which are only hydrogen bonded to the grafted strand and can therefore be denatured and washed away).

I very much like the idea to change the recipe so that the dark cycles are imaged ensuring we capture the entire index2 read. Tech Support is not very forthcoming on editing HiSeq recipes, but if this happens to us again I will try this solution and update this thread with the protocol and outcome.

As it turns out, in this case we could demultiplex with only index1 and 2 bases from index2.

Thanks again!